The long-term objective of the proposed project is to identify the antigen(s) expressed by human melanoma cells that serve as epitope(s) for melanoma-specific cytotoxic T-lymphocytes (CTL). The clinical implications of the identification and synthesis of these antigens include (1) the possibility of creating a safe recombinant vaccine for melanoma, (2) the possibility of stimulating human lymphocytes in vitro for adoptive transfer to melanoma patients, using a specific stimulant of melanoma-specific CTL, and (3) the possibility of identifying the proteins) and genetic material from which these antigens are derived, which would in itself have implications for understanding malignant transformation of melanoma. HLA-A2 antigens will be purified from the cell surface and cytoplasm of a least 10(9) HLA-A2(+) human melanoma cells. Preparations serving as controls will be purified from A2(+) non-melanoma cells. The peptides bound in the cleft of the A2 molecules will be eluted by acidification and filtration centrifugation. The resulting peptide mixtures will be analyzed by mass spectrometry under electrospray ionization conditions. Peptides identified by mass spectrometry that are present on melanoma cells but absent on non-melanoma cells will be sequenced by mass spectrometric methods. The sequenced peptides will be synthesized and tested for their ability to render A2(+) non-melanomas sensitive to lysis by HLA-A2-restricted melanoma-specific cytotoxic T lymphocytes.